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WO2018187482A1 - Procédés de production de peptides lasso biologiquement actifs - Google Patents

Procédés de production de peptides lasso biologiquement actifs Download PDF

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WO2018187482A1
WO2018187482A1 PCT/US2018/026101 US2018026101W WO2018187482A1 WO 2018187482 A1 WO2018187482 A1 WO 2018187482A1 US 2018026101 W US2018026101 W US 2018026101W WO 2018187482 A1 WO2018187482 A1 WO 2018187482A1
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lasso
peptide
nucleic acid
peptides
display library
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Douglas Alan MITCHELL
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University of Illinois at Urbana Champaign
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
    • C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/76—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Actinomyces; for Streptomyces
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
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    • C12N2330/00—Production
    • C12N2330/50—Biochemical production, i.e. in a transformed host cell

Definitions

  • Lasso peptides are ribosomaily assembled and post-translationaily modified peptides (RiPPs) that are produced by bacteria. Lasso peptides are some of the smallest possible globular proteins (Fig, 1A). However, the unique topology renders the fold inaccessible to chemical synthesis but remarkably stable towards heat and proteases. Combined with their naturally diverse and expansive surfaces relative to small molecules, lasso peptides are an ideal starting point to inhibit (or associate) targets deemed undruggable by small molecules.
  • Lasso peptides are difficult to recombinantly or synthetically produce.
  • Thermophilic and actinomycete mature lasso peptides and lasso peptides of the biosynthetic gene cluster are particularly difficult to produce in the laboratory.
  • Methods are needed in the art to produce biologically active, soluble, and stable mature lasso peptides and biologically active, soluble, and stable lasso peptides of the synthetic gene duster.
  • An embodiment provides methods of producing a mature lasso peptide.
  • the methods comprise transforming a host ceil with a first plasmid comprising a nucleic acid molecule encoding a lasso precursor peptide operabiy linked to a solubility enhancing polypeptide and a second plasmid comprising a nucleic acid molecule encoding a lasso leader peptidase; a nucleic acid molecule encoding a lasso cyclase; and a nucleic acid molecule encoding a RiPP recognition element (RRE) to generate a transformed host cell.
  • the transformed host cell can be cultured in media.
  • the mature lasso peptide can be extracted from the host cell or the culture media.
  • the lasso peptide can be produced at a yield of more than 0.5 mg/L of culture media.
  • the nucleic acid molecule encoding a lasso precursor peptide can be an actinomycete or thermophiie nucleic acid molecule.
  • the nucleic acid molecule encoding a lasso leader peptidase can be an actinomycete or thermophiie nucleic acid molecule.
  • the nucleic acid molecule encoding a lasso cyclase can be an actinomycete or thermophiie nucleic acid molecule.
  • the nucleic acid molecule i encoding a RiPP recognition element (RRE) can be an actinomycete or thermophile nucleic acid molecule.
  • the host cell can be a mesophile.
  • the mature lasso peptide can be an actinomycete lasso peptide or a thermophile lasso peptide.
  • Another embodiment provides methods of producing one or more peptides of a lasso biosynthetic gene duster.
  • the methods can comprise transforming a host cell with (i) a plasmid comprising a nucleic acid molecule encoding an actinomycete lasso cyclase or a thermophile lasso cyclase operably linked to a solubility enhancing polypeptide; (ii) a plasmid comprising a nucleic acid molecule encoding a thermophile lasso leader peptidase or an actinomycete lasso leader peptidase operably linked to a solubility enhancing polypeptide; (iii) a plasmid comprising a nucleic acid molecule encoding a thermophile lasso RiPP recognition element or an actinomycete lasso RiPP recognition element operably linked to a solubility enhancing polypeptide; (iv) a plasmid comprising
  • Still another embodiment provides methods of producing a mature lasso peptide in vitro comprising combining one or more purified recombinant actinomycete or thermophile lasso cyclases, one or more purified recombinant actinomycete or thermophile lasso leader peptidases, one or more purified recombinant actinomycete or thermophile lasso RiPP recognition elements, and one or more purified recombinant actinomycete or thermophile lasso precursor peptides) in vitro under conditions suitable for lasso peptide formation, such that a mature lasso peptide is produced.
  • the lasso peptide can be produced at a yield of more than 1 mg/L.
  • Another embodiment provides a method of producing a mature lasso peptide in vitro comprising combining one or more purified recombinant lasso precursor peptides lacking a leader portion, one or more purified recombinant lasso cyclases, and adenosine triphosphate (ATP) in vitro under conditions suitable for lasso peptide formation, such that a mature lasso peptide is produced.
  • the lasso precursor peptide lacking a leader portion can be an actlnomycete or thermophile peptide.
  • the lasso cyclase can be an actlnomycete or thermophile peptide.
  • Yet another embodiment provides methods of screening for biological activity of a lasso peptide.
  • the method comprises displaying one or more tagged lasso precursor peptides in a display library.
  • the display library is contacted with a purified lasso cyclase, a purified lasso leader peptidase, and a purified lasso RiPP recognition element to form a lasso peptide display library.
  • the lasso peptide display library can be with one or more test agents.
  • the display library can be a phage display library, a phagemid display library, a virus display library, a bacterial cell display library, yeast display library, a Agt1 1 library, an in vitro library selection system (CIS display), an in vitro compartmentalization library, an antibody- ribosome-mRNA (ARM) ribosome display library, or a ribosome display library.
  • the one or more tagged lasso precursor peptides can be mesophile, actlnomycete, or thermophile peptides.
  • the lasso cyclase, the lasso leader peptidase, and the lasso RiPP recognition element can be actinomycete or thermophile peptides.
  • the display library can be a phage display library, a phagemid display library, a virus display library, a bacterial ceil display library, yeast display library, a Agt1 1 library, an in vitro library selection system (CIS display), an in vitro compartmentalization library, an antibody-ribosome-mRNA (ARM) ribosome display library, or a ribosome display library.
  • the mature lasso peptides can be actinomycetes mature lasso peptides or thermophile mature lasso peptides.
  • FIG. 1 Panels A-B.
  • A Space-filling model of a typical lasso peptide, acinetodin (PDB 5UI6), highlighting the globular shape and dimensions.
  • B Lasso peptide BGC with essential genes highlighted.
  • the gene nomenclature and coloring adopted throughout is: A, precursor peptide (black); B, leader peptidase (homologous to transglutaminase, orange); C, lasso cyclase (homologous to asparagine synthetase, blue); £, RRE (RiPP Recognition Element, homologous to PqqD, yellow).
  • Precursors are comprised of leader and core regions. After leader removal, the cyclase is presumed to adenylate Asp/Glu. The substrate must be "pre- folded" prior to macrocyclization given the known steric restraints
  • Fig, 2 RODEO workflow.
  • the RODEO algorithm takes a list of protein accessions as input and retrieves a user-defined number of flanking genes from GenBank. These genes are then functionally analyzed. Lastly, RODEO performs a 6-frame translation of intergenic regions. These peptides are then scored to identify the most likely lasso precursor. See: ripprodeo.org for more information.
  • Fig. 3 Pane!s
  • A-E RODEO-esiabied insights into lasso peptides.
  • A Distribution of BGCs amongst bacteria.
  • B BGCs of two anantins (previously unknown origin) and four novel lassos. Naming and color-coding identical to Fig. 1 .
  • C Precursor sequences corresponding to panel (B).
  • D Topologies of lasso peptides. Frequencies for class l-IV are 1 .4%, 96%, 2%, 0.6%, respectively.
  • E Solution structure of LP2006: a rare disuifide-containing, class IV lasso peptide (SEQ ID NO:6).
  • Fig. 4 Panels A-E. Ther obifida lasso peptides.
  • A Sequences of TfusA (SEQ ID NO:7)/TcelA (SEQ ID NO:8)(BGC architecture identical to Fig 3B).
  • B MS- confirmed E. coil production of fusilassin.
  • C Simplified structural representation of fusilassin.
  • D SDS-PAGE of MBP-tagged Tcel and Tfus proteins (unoptimized expression, single-column purification). Yields for TcelABCE and TfusABCE, respectively: 5, 14, 8, 50 mg/L and 8, 20, 3, 32 mg/L.
  • Fig, 5 shows the structure of pACYC-TfusC-TFusEB
  • Fig. 6 shows heterologous expression/biosynthesis of fusilassin in E. coii.
  • Fig. 7 shows a general structure of a lasso peptide.
  • Fig. 8 shows an example of a two p!asmid construct for expression of a lasso peptide by a host cell.
  • Fig. 9 shows an alternate construct for expression of a lasso peptide by a host ceil.
  • Fig. 10 shows an alternate construct for expression of a lasso peptide by a host cell.
  • Fig, 11 shows ALDI-TOF-MS data showing in vivo production of fusilassin biosynthesis (m/z 2269) and in vivo production of position 1 mutants of fusilassin. Position 1 of the core peptide was substituted with W (m/z 2289), H (m/z 2220), K (m/z 221 1 ), L (m/z 2196), or A (m/z 2154).
  • XYTAEWGLELI FVFPRFI is SEQ I D NO:20.
  • compositions and methods are more particularly described below and the Examples set forth herein are intended as illustrative only, as numerous modifications and variations therein will be apparent to those skilled in the art.
  • the meaning of "a”, “an”, and “the” includes plural reference unless the context clearly dictates otherwise.
  • the term “about” in association with a numerical value means that the value varies up or down by 5%. For example, for a value of about 100, means 95 to 105 (or any value between 95 and 105).
  • Lasso peptides are ribosomaily synthesized and post-translationally modified peptidic (RiPP) products.
  • RiPP biosynthesis begins with the processing of a gene- encoded precursor peptide, which is comprised of functionally distinct leader and core portions (Fig. 1 B).
  • the leader harbors a unique motif, known as the recognition sequence, that specifically recruits biosynthetic proteins while the core region receives ail of the chemical modifications. After modification, the leader is proteolytically removed.
  • the biosynthetic enzymes can be specific for a particular recognition sequence yet promiscuously process diverse, even unrelated, core sequences.
  • RiPPs require minimal genomic space, with many lasso peptide biosynthetic gene clusters (BGCs) needing ⁇ 3 kb.
  • BGCs lasso peptide biosynthetic gene clusters
  • the lasso topology imparts notable stability. While lasso peptide biosynthesis is known is general terms, there is little molecular insight into how lasso peptides are actually formed. Lasso peptides occupy a chemical and functional space between small molecules and proteins. Indeed, rather than behaving like a polypeptide, the traits of a lasso peptide are more consistent with a small molecule. In support of this observation, lasso peptides have therapeutic activity after oral administration, which conflicts with a long-standing dogma regarding peptidic drugs.
  • lasso peptide bioactivities include antagonism of various cell- surface proteins, including the receptors for atrial natriuretic peptide, glucagon, and endotheiin.
  • the lasso peptide siamycin interacts with HIV envelope proteins and prevents viral fusion to CD4 + T cells.
  • Enzymes can also be lasso peptide targets, as illustrated by lassomycin, which selectively blocks mycobacterial ClpC1 protease, as well as microcin J25 and capistruin which inhibit RNA polymerase after employing a Trojan horse-like strategy to gain entry into Gram-negative proteobacteria.
  • acrocyciic peptides with notable bioactivities include influenza virus fusion inhibitors, PD-1 /PD-L1 inhibitors for anticancer applications, insulin-degrading enzyme inhibitors for type-2 diabetes, etc.
  • influenza virus fusion inhibitors PD-1 /PD-L1 inhibitors for anticancer applications
  • insulin-degrading enzyme inhibitors for type-2 diabetes, etc.
  • Lasso peptides recapitulate the desired properties of synthetic cyclic peptides (e.g.
  • siamycin also blocks viral fusion) but with two major advantages: (I) lasso peptides are entirely genetically encoded and (II) the presence of a free C- terminal tail in the lasso provides an anchor point for surface display or other high- throughput screening methods.
  • Rapid ORF Description & Evaluation Online can be used to identify lasso gene clusters. See, Tietz et a!,, A new genome-mining tool redefines the lasso peptide biosynthetic landscape. Nature Chemical Biology, 13:470TM 478 (2017), which is incorporated herein by references in its entirety.
  • This program profiles genes neighboring a query and automates the genome-mining process (Fig. 2).
  • RODEO is distinguished from other programs (e.g. antiS ASH and PRISM) by taking a protein-centric perspective to genome-mining (i.e. all lasso peptide gene clusters can be queried in a single run rather than one query per genome). RODEO accurately predicts several classes of RiPP precursor peptides.
  • Lasso peptide biosynthesis remains largely enigmatic. This lack of understanding is a function of the difficulties in identifying lasso peptide gene clusters prior to the development of RODEO and the poor stability/solubility of purified lasso peptide biosynthetic enzymes.
  • RODEO can be used to assist in identifying lasso peptide gene clusters (Fig,
  • a mature lasso peptide is a post-fransiationaliy modified, biologically active molecule as shown in, e.g., Fig. 1 B, 3D, and 8.
  • Lasso peptides comprise about 20 amino acids, with an average size range of about 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 amino acids, with molecular weights of about 1 S0Q--25QG Da).
  • Lasso peptides comprise a ring of about 7, 8, or 9 amino acids, which can be closed by a lactam bond between the N-terminai amino group and the carboxyiate side chain of a glutamate or an aspartate.
  • the tail can be trapped within the ring either by bulky side chains (steric trapping) or by one or two disulfide bonds, or by both means.
  • This specific lasso (or lariat) topology makes lasso peptides extraordinarily stable. See Figure 7.
  • Methods of recombinantly producing mature lasso peptides comprise transforming a host cell with a first plasmid or a first nucleic acid molecule comprising a nucleic acid molecule encoding a lasso precursor peptide operabiy linked to a solubility enhancing polypeptide and a second plasmid or second nucleic acid molecule comprising a nucleic acid molecule encoding a lasso leader peptidase; a nucleic acid molecule encoding a lasso cyclase; and a nucleic acid molecule encoding a RiPP recognition element (RRE) to generate a transformed host cell.
  • RRE RiPP recognition element
  • a solubility enhancing polypeptide or tag is a polypeptide that is operabiy linked to a protein (e.g., lasso precursor peptide, lasso leader peptidase; lasso cyclase; a RiPP recognition element (RRE)), and which can help to properly fold the protein of interest leading to enhanced solubility of the protein of interest.
  • concentration, total yield, solubility, biological activity, or combinations thereof are about 5, 10, 15, 20, 25, 30, 40, 50, 80, 75% or more improved for the protein of interest when a solubility enhancing polypeptide or fag is used as compared to the concentration, total yield, solubility, biological activity, or combinations thereof where a solubility enhancing polypeptide is not used.
  • solubility enhancing polypeptides include maltose binding protein ("MBP") (Lebendiker & Daudgei, Purification of proteins fused to maltose-binding protein, Methods Moi. Biol. 681 :281 (201 1 )), thioredoxin, transcription elongation factor NusA, thiol-disulfide oxidoreductase, glutathione S-transferase (GST), protein G B1 domain, protein D, the Z domain of Staphylococcal protein A, GB1 asic , Calmodulin, Poly Arg or Lys peptide tags, SUMO small ubiquitin-modifier, synthetic solubility- enhancing tags, DsbC Disufide bond C, Skp seventeen kilodaiton protein, T7PK Phage T7 protein kinase, and ZZ (protein A IgG ZZ repeat domain).
  • MBP maltose binding protein
  • ZZ protein A IgG Z
  • a solubility enhancing polypeptide or tag can be linked or fused to an N- terminus or a C-terminus of the protein of interest.
  • the transformed host cell is cultured in a culture media such that mature lasso peptides that are post-translationaliy modified and biologically active are produced.
  • the lasso peptides can be extracted or isolated from the host ceil or the culture media.
  • the second plasmid (which comprises one or more nucleic acid molecules encoding a lasso leader peptidase; one or more nucleic acid molecules encoding a lasso cyclase; and one or more nucleic acid molecules encoding a RiPP recognition element (RRE)) can be replaced with multiple plasmids.
  • nucleic acid molecules encoding a lasso leader peptidase; one or more of the nucleic acid molecules encoding a lasso cyclase; and one or more of the nucleic acid molecules encoding a RiPP recognition element (RRE) can each be present on a single plasmid or a combination of two or more of the nucleic acids can be present on a single plasmid.
  • the one or more nucleic acid molecules encoding a lasso leader peptidase; one or more nucleic acid molecules encoding a lasso cyclase; and one or more nucleic acid molecules encoding a RiPP recognition element (RRE)) are operably linked to a solubility enhancing polypeptide.
  • a recombinantly produced, mature Iasso peptide can be produced at a yield of about 0.5, 0.75, 1 .0, 1 .5, 2.0, 3.0, 4.0, 5.0 mg/L or more of culture media,
  • a recombinantly produced mature Iasso peptide has about 90, 95, 100, 105, or 1 10%, of the biological activity of a corresponding natural or wild-type Iasso peptide.
  • a recombinantly produced mature iasso peptide has about 90, 95, 100, 105, or 1 10%, of the proteolytic stability of a corresponding natural or wild-type Iasso peptide
  • a recombinantly produced mature !asso peptide has about
  • a recombinantly produced mature iasso peptide has about 90, 95, 100, 105, or 1 10%, of the solubility of a corresponding natural or wild-type Iasso peptide.
  • the Iasso peptides are from an actinomycete, thermophile, or mesophile
  • the host cell is a bacterium from the order Enterobacteriaies, such as Escherichia coii.
  • the host cell is a bacterium from the family Enterobacteriaceae.
  • the host cell is from the Firmicufes phylum, including for example, Bacillus sp., or Bacillus subtiiis.
  • the host cell is a Sinorhizobium sp., such as Sinorhizobium meiiloti, Sinorhizobium medicae, and Sinorhizobium fredii.
  • Mature Iasso peptides, Iasso precursor peptides, iasso leader peptidases, Iasso cyclases, and RiPP recognition element can be actinomycete, thermophile, or mesophile peptides.
  • the mature Iasso peptide, precursor peptide, Iasso leader peptidase, iasso cyclase, or RiPP recognition element has about 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100% homology to a naturally occurring actinomycete, thermophile, or mesophile mature Iasso peptide, iasso precursor peptide, iasso leader peptidase, iasso cyclase, or RiPP recognition element.
  • a mature iasso peptide, precursor peptide, iasso leader peptidase, Iasso cyclase, or RiPP recognition element has about 50, 60, 70, 80, 90% or more homology to a naturally occurring actinomycete, thermophile, or mesophile mature Iasso peptide, Iasso precursor peptide, iasso leader peptidase, Iasso cyclase, or RiPP recognition element, in an embodiment a mature iasso peptide, precursor peptide, lasso leader peptidase, lasso cyclase, or RiPP recognition element can be a synthetic or a mutant lasso polypeptide.
  • Nucleic acid molecules that encode lasso precursor peptides, lasso leader peptidases, lasso cyclases, and RiPP recognition element can be actinomycete, thermophiie, or mesophiie nucleic acid molecules.
  • nucleic acid molecules encoding the lasso precursor peptides, lasso leader peptidase, lasso cyclase, or RiPP recognition element have about 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100% homology to naturally occurring actinomycete, thermophiie, or mesophiie nucleic acid molecules that encode lasso precursor peptides, lasso leader peptidases, lasso cyclases, or RiPP recognition elements.
  • nucleic acid molecules that encode precursor peptides, lasso leader peptidases, lasso cyclases, or RiPP recognition elements have about 50, 60, 70, 80, 90% or more homology to a naturally occurring actinomycete, thermophiie, or mesophiie nucleic acid molecules that encode lasso precursor peptides, lasso leader peptidases, lasso cyclases, or RiPP recognition elements.
  • nucleic acid molecules encoding a precursor peptide, lasso leader peptidase, lasso cyclase, or RiPP recognition element can be a synthetic nucleic acid or a mutant lasso nucleic acid.
  • Actinomycetes are members of the phylum Actinobacteria. Actinomycetes include, for example, Actinomycetaceae, Actinopoiysporaceae, Catenuiisporaceae, Actinospicaceae, Corynebacteriaceae, Dietziaceae, Gordoniaceae, Mycobacteriaceae, Nocardiaceae, Segniliparaceae, Tsukamureilaceae, Acidothermaceae, Cryptosporangiaceae, Frankiaceae, Geodermatophilaceae, Nakamure!!aceae, Sporichthyaceae, Glycomycetaceae, Jiangeilaceae, Kineosporiaceae, Beutenbergiaceae, Bogorieilaceae, Brevibacte iaceae, Celiuiomonadaceae, Demequinaceae, Dermabacteraceae, Dermatophilacea
  • Actinomycete lasso peptides include but are not limited to, for example, fusilassin from Thermobifida fusca, sphaericin from P!anomonospora sphaerica; streptomonomicin from Streptomonospora alba; raynimysin from Streptomyces; gelsomycin from Streptomyces; adanomysin from Streptomyces; anantin from Streptomyces; frankimysin from Frankia; g!ycimysin from Actinomyces; AnantinBi from Streptomyces sp, NRRL-S-146; AnantinBa from Streptomyces sp, NRRL-S-146; Anantin C from Streptomyces olindensis; citrulassin A from Streptomyces aurantiacus NRRL B-3066; citrulassin B from Streptomyces
  • NRRL S-146 citrulassin D from Streptomyces katrae NRRL B-16271 ; citrulassin E from Streptomyces giaucescens NRRL ISP-5155, Streptomyces giaucescens NRRL B- 2899, Streptomyces giaucescens NRRL B-1 1408, or Streptomyces giaucescens NRRL B-2708; Citrulassin F from Streptomyces avermitilis NRRL B-16169; Keywimysin from Streptomyces sp. NRRL F-5702, Streptomyces sp.
  • NRRL F-5681 or Streptomyces sp. NRRL F-2202; lagmysin form Streptomyces sp. NRRL S-1 18; LP2006 from Nocardiopsis aiba; moomysin from Streptomyces cattieya sp. NRRL 8057; anantin B form Streptomyces sp. NRRL S-146; anantin C from Streptomyces oiindensis, citrulassin A from Streptomyces aibuius NRRL B-3066; citrulassin B from Streptomyces aurantiacus NRRL B-2806; citrulassin C from Streptomyces sp.
  • NRRL S-146 citrulassin D from Streptomyces katrae NRRL B-16271 ; citrulassin E from Streptomyces giaucescens NRRL ISP-5155; citrulassin F from Streptomyces avermitilis NRRL B-16169; keywimysin from Streptomyces sp. NRRL; F-5681 ; keywimysin from Streptomyces sp. NRRLF-2202; keywimysin Streptomyces sp. NRRL F-5702; lagmysin Streptomyces sp.
  • Thermophiles can be fungi, archaea, or bacteria that have an optimum growth temperature of about 50° or more, a maximum of up to about 70°C or more, and a minimum of about 20°C.
  • Thermophilic fungi include, for example, Acremonium, Aureobasidium, Cryptococcus, Filibasidium, Fusarium, Humicoia, Magnaporthe, Mucor, Myceiiopbthora, Neurospora, Paecilomyces, Peniciliium, Schizophyilum, Talaromyces, Thermoascus, Thielavia, and Tolypociadium.
  • Thermophilic microorganisms are eubacteria or archaebacteria and include for example the following genera: Thermus, Bacillus, Thermococcus, Pyrococcus, Aeropyrum, Aquifex, Suifolobus, Pyrolobus, or Methanopyrus.
  • Specifc examples include Thermobispora bispora, Thermobacculum terrenum, Thermobifida fusca, Thermobifida DCiuiosiiytica, Thermoactinomyces vulgaris, Thermus aquaticus, Thermus thermophilus, Bacillus stearothermophilus, Aquifex pyrophi!us, Geothermobacterium ferrireducens, Thermotoga maritime, Thermotoga neopoiitana, Thermotoga petrophila, Thermotoga naphthophila, Acidianus Infemus, Aeropyrum pernix, Archaeogiobus fulgidus, Archaeoglobus profundus, Caldivirga maquiiingensis, Chloroflexus aurantiacus, Desulfurococcus amylolyticus, Desulfurococcus mobilis, Desulfurococcus mucos
  • Mesophiles are microorganisms thai grows best in moderate temperatures between about 20 and 50°C. Mesophiles can be archaea, bacteria or fungi.
  • a mesophile, thermophile, or actinomycete lasso leader peptidase, lasso cyclase, lasso precursor peptide, lasso RiPP recognition element, or combination thereof can be produced.
  • a host cell can be transformed with (1 ) a piasmid comprising a nucleic acid molecule encoding a mesophile, thermophile, or actinomycete lasso leader peptidase optionally operably linked to a solubility enhancing polypeptide; (2) a p!asmid comprising a nucleic acid molecule encoding a mesophile, thermophile, or actinomycete lasso cyclase optionally operably linked to a solubility enhancing polypeptide; (3) a piasmid comprising a nucleic acid molecule encoding a mesophile, thermophile, or actinomycete lasso precursor peptide optionally operably linked to a so
  • the transformed host cell can be cultured in a culture medium.
  • the lasso leader peptidase, lasso cyclase, lasso precursor peptide, lasso RiPP recognition element, or combination thereof can be extracted from the host cell or the culture media.
  • lasso leader peptidase, lasso cyclase, lasso precursor peptide, or lasso RiPP recognition element can be produced at a yield of about 0.5
  • a solubility enhancing peptide can be cleaved from a lasso leader peptidase, lasso cyclase, lasso precursor peptide, or lasso RiPP recognition element using a protease,
  • a recombinantly produced lasso leader peptidase, lasso cyclase, lasso precursor peptide, lasso RiPP recognition element has about 90,
  • lasso RiPP recognition element 95, 100, 105, 1 10%, or more biological activity of a corresponding natural or wild-type lasso leader peptidase, lasso cyclase, lasso precursor peptide, or lasso RiPP recognition element.
  • a recombinantly produced lasso leader peptidase, lasso cyclase, lasso precursor peptide, lasso RiPP recognition element has about 90, 95, 100, 105, or 1 10%, of the proteolytic stability of a corresponding natural or wild-type lasso leader peptidase, lasso cyclase, lasso precursor peptide, or lasso RiPP recognition element,
  • a recombinantly produced lasso leader peptidase, lasso cyclase, lasso precursor peptide, lasso RiPP recognition element has about 90, 95, 100, 105, or 1 10%, of the thermal stability of a corresponding natural or wild-type lasso leader peptidase, lasso cyclase, lasso precursor peptide, or lasso RiPP recognition element.
  • a recombinantly produced lasso leader peptidase, lasso cyclase, lasso precursor peptide, lasso RiPP recognition element has about 90, 95, 100, 105, or 1 10%, of the solubility of a corresponding natural or wild-type lasso leader peptidase, lasso cyclase, lasso precursor peptide, lasso RiPP recognition element.
  • a recombinantly produced lasso RiPP recognition element can bind a leader of a lasso precursor peptide with an affinity for a lasso leader peptide of about 30, 40, 50, 80, 70, 80, or more nM.
  • Mature lasso peptides can be reconstituted in vitro (also called total biosynthesis).
  • one or more purified recombinantly produced lasso leader peptidases, one or more purified recombinantly produced lasso cyclases, one or more purified recombinantly produced lasso precursor peptides, and one or more purified recombinantly produced lasso RiPP recognition elements are combined in vitro under suitable conditions to produce a lasso peptide.
  • a lasso peptide can be produced at a yield of about 0.5, 1 , 2, 3, 4, 5, 6 mg/L or more.
  • a lasso peptide can be produced in vitro by combining one or more purified recombinantly produced lasso precursor peptides, one or more purified recombinantly produced lasso cyclases, and adenosine triphosphate (ATP) in vitro under conditions suitable for lasso peptide formation, such that a mature lasso peptide is produced.
  • adenosine triphosphate adenosine triphosphate
  • a mature lasso peptide in vitro by combining one or more purified recombinant lasso precursor peptides lacking a leader, one or more recombinantly produced lasso cyclases, and adenosine triphosphate (ATP) in vitro under conditions suitable for lasso peptide formation, such that a mature lasso peptide is produced.
  • a lasso precursor peptide is comprised of a leader and a core. For this method, the leader portion of the lasso precursor peptide is not necessary.
  • the lasso precursor peptide can be cleaved (using e.g., a protease) such that the leader is removed and the core portion used in the method.
  • a lasso precursor peptide lacking a leader can be produced recombinantly by transforming a host ceil with a polynucleotide encoding only the core portion of a lasso precursor peptide. This core peptide can then be purified and used in the method.
  • a lasso precursor peptide lacking a leader portion is missing about 8, 10, 12, 15, 20, 30 or more amino acids of the leader portion
  • a lasso precursor peptide lacking a leader portion is missing all of the leader portion amino acids.
  • a lasso peptide can be produced at a yield of about 0.5, 1 , 2, 3, 4, 5, 6 mg/L or more.
  • the core sequences of lasso precursor peptides are known to those of skill in the art, and can be found in, for example Tietz et a/. . , Nat. Chem. Biol. 13:470 (2017), see supplementary Table 8.
  • In vitro reconstituted mature lasso peptides can have about 50, 60, 70, 80, 90, 100, 1 10 %, or more biological activity as compared to a natural or wild-type lasso peptide.
  • In vitro reconstituted mature lasso peptides can have about 50, 60, 70, 80, 90, 100, 1 10%, or more proteolytic stability as compared to a natural or wild-type lasso peptide
  • In vitro reconstituted mature lasso peptides can have about 50, 60, 70, 80, 90,
  • thermal stability as compared to a natural or wild-type lasso peptide.
  • in vitro reconstituted mature lasso peptides can have about 50, 60, 70, 80, 90, 100, 1 10%, or more solubility as compared to a natural or wild-type lasso peptide.
  • the in vitro reconstituted mature lasso peptides are derived from an actinomycete or thermophiie and the host cell is a bacterium from the order Enterobacteriaies, such as Escherichia coii.
  • the host cell is a bacterium from the family Enterobacteriaceae.
  • An embodiment provides methods of screening for activity of a lasso peptide.
  • One or more lasso precursor peptides can be displayed in a display library.
  • the lasso precursor peptide can be displayed as a fusion to a protein tag, such as Aga2p. See Boder & Wittrup, Nat. Biotechnol. 1997; 15:553.
  • the lasso precursor peptide is displayed on the surface of library, by for example the C terminus. This is a lasso precursor peptide display library.
  • the lasso precursor peptide display library can be contacted with one or more of a purified lasso cyclase, a purified lasso leader peptidase, and a purified lasso RiPP recognition element to form a lasso peptide display library.
  • mature lasso peptides are displayed on the surface of the display library to form a mature lasso peptide display library.
  • the enzymes and proteins used in this method can be recombinantiy produced. It is important that the purified lasso cyclase, lasso leader peptidase, and/or lasso RiPP recognition element are stable and have robust activity in order to form the mature lasso peptide display library.
  • the purified lasso cyclase, lasso leader peptidase, and/or lasso RiPP recognition element can be recombinant peptides produced by methods described herein.
  • the lasso cyclase, lasso leader peptidase, and/or lasso RiPP recognition element can be operably linked to a solubility enhancing polypeptide.
  • a lasso precursor peptide display library or mature lasso peptide display library can comprise a phage display library.
  • a phage display library can be a collection of phage that have been genetically engineered to express one or more lasso precursor peptides on their outer surface.
  • nucleic acid molecules encoding the lasso precursor peptides are inserted in frame into a gene encoding a phage capsule protein, in another embodiment, a phage display library is a collection of phage that display one or more mature lasso peptides on their outer surface.
  • a display library can be, for example, a phage display library, a phagemid display library, a virus display library, a bacterial ceil display library, yeast display library, a Agt1 1 library, an in vitro library selection system (CIS display), an in vitro compartmentalization library, an antibody- ⁇ ribosome ⁇ mRNA (ARM) ribosome display library, or a ribosome display library.
  • a mature lasso peptide display library can be screened for biological activity such as anti-microbial activity, receptor antagonist activity (e.g. , endotheiin receptor, natriuretic system, glucagon receptor), enzyme inhibitor activity (e.g. , inhibitor of smooth muscle myosin light chain kinase (MLCK), prolyioligopeptidase, RNA polymerase), and inhibitor activity of, for example, H IV. Therefore, test agents such as bacteria, fungi, viruses, prokaryotic or eukaryotic cells, drugs, cellular receptor proteins, proteins, nucleic acids, enzymes, and small molecules can be added to a mature lasso peptide display library.
  • the mature lasso peptides can be mesophile mature lasso peptides, actinomycetes mature lasso peptides, or thermophiie mature lasso peptides.
  • the lasso peptide display library can then be contacted with one or more test agents.
  • the effect of the lasso peptides on the test agent can then be determined.
  • Lasso peptides can have a wide variety of biological activities including, for example, inhibition of binding, inhibition of enzyme activity, bactericidal activity, bacteriostatic activity, virucidal activity, and virustatic activity.
  • a screening step can also serve as the step of recovering a test agent that binds to the mature lasso peptide.
  • Solid-phase screening methods can involve, for example, immobilizing target agents onto a solid phase, and contacting lasso peptides contained in a liquid phase with the target agents, removing unbound lasso peptides nonspecifically bound lasso peptides, and then selectively separating lasso peptides bound with the target agent to screen for a peptide having, for example, a desired binding activity.
  • a liquid-phase screening method can involve, for example, contacting lasso peptides with target agents in a solution, removing unbound lasso peptides and nonspecifically bound lasso peptides, and then selectively separating the lasso peptides bound with target agents.
  • a recombinant host ceil, transgenic host cell, or transformed host ceil is a cell into which one or more foreign or exogenous nucleic acid molecules, synthetic nucleic acid molecules, or plasmids have been introduced or inserted into the ceil.
  • the one or more foreign nucleic acid molecules, synthetic nucleic acid molecules, or plasmids do not occur in the host cell in nature.
  • An exogenous or foreign nucleic acid molecule can be from a different species (and so heterologous), or from the same species (and so homologous), relative to the cell being transformed.
  • An exogenous gene can include a homologous gene that occupies a different location in the genome of the cell or is under different control, relative to the endogenous copy of the gene.
  • An exogenous gene may be present in more than one copy in the cell.
  • An exogenous gene can be maintained in a cell as an insertion into the genome or as an extrachromosomal molecule.
  • Suitable host ceils for expression of nucleic acid molecules are microbial cells that can be found broadly within the fungal or bacterial families and that grow over a wide range of temperature, pH values, and solvent tolerances.
  • suitable host strains include but are not limited to fungal or yeast species, such as Aspergillus, Trlchoderma, Saccharomyces, Pichla, Candida, Hansenuia, and Kluyvero yces, or bacterial species, such as member of the proteobacteria and actinomycetes as well as the specific genera Acinetobacter, Arthrobacter, Brevibacterium, Acidovorax, Bacillus, Clostridia, Streptomyces, Escherichia (e.g., E. coll), Salmonella, Pseudomonas, and Cornyebacterium.
  • a host cell can be a mesophile, thermophile, or actinomycete ceil.
  • Transformation is a process of introducing foreign or exogenous genetic material into a host cell.
  • An isolated nucleic acid molecule, peptide, or polypeptide refers to nucleic acid molecule, peptide, or polypeptide that is separated from at least one contaminant with which it is ordinarily associated in its source. Thus, an isolated nucleic acid molecule, isolated peptide, or isolated polypeptide is present in a form or setting that is different from that in which it is found in nature.
  • An isolated nucleic acid molecule can be a naturally-occurring polynucleotide that is not immediately contiguous with one or both of the 5 ! and 3' flanking genomic sequences that it is naturally associated with.
  • a nucleic acid molecule or polynucleotide is a nucleic acid molecule (e.g., DNA or RNA) that can comprise coding sequences necessary for the production of a peptide, polypeptide, or protein precursor.
  • the encoded polypeptide may be a full- length polypeptide, a fragment thereof (less than full-length), or a fusion of either the full-length polypeptide or fragment thereof with another polypeptide, yielding a fusion polypeptide.
  • a peptide, protein, or polypeptide is any chain of amino acids, regardless of length or post-transiational modification (e.g., glycosylation or phosphorylation).
  • An expression vector, p!asmid, or recombinant DNA construct is a vehicle for introducing one or more nucleic acid molecules into a host ceil.
  • the nucleic acid molecule can be one that has been generated via human intervention, including by recombinant means or direct chemical synthesis.
  • the nucleic acid molecule can include one or more nucleic acid elements that permit transcription and/or translation of a particular nucleic acid molecule.
  • An expression vector can be part of a plasmid, virus, or nucleic acid fragment, or other suitable vehicle.
  • An expression vector can include, for example, a nucleic acid to be transcribed operably linked to a promoter.
  • Operably linked means two or more nucleic acid molecules that are functionally linked together, such as one or more control sequences (e.g., a promoter) and one or more target nucleic acid molecules (e.g., molecules that encodes a protein) or two or more target nucleic acid molecules that are linked. Where two or more target nucleic acid molecules are linked the result can be a fusion protein.
  • a promoter is operably linked to a target nucleic acid molecule where it can mediate transcription of the target nucleic acid molecule.
  • a purified polypeptide or peptide is a polypeptide or peptide preparation that is substantially free of cellular material, other types of polypeptides, peptides, chemical precursors, chemicals used in synthesis of the polypeptide or peptide, or combinations thereof.
  • a polypeptide or peptide preparation that is substantially free of cellular material, culture medium, chemical precursors, chemicals used in synthesis of the polypeptide or peptide, etc. has less than about 30%, 20%, 10%, 5%, 1 % or more of other polypeptides or peptides, culture medium, chemical precursors, and/or other chemicals used in synthesis. Therefore, a purified polypeptide or peptide is about 70%, 80%, 90%, 95%, 99% or more pure.
  • a purified polypeptide or peptide does not include unpurified or semi-purified cell extracts or mixtures of polypeptides that are less than 70% pure.
  • Lasso peptide biosynthetic gene clusters from thermophiles were identified in an attempt to provide more stable and soluble lasso enzymes.
  • Biosynthetic gene clusters from Thermobispona bispora, Thermobifida fusca (“Tfus”), and Thermobifida DCluiosilytica (“Tcei”) were identified as targets.
  • An unusual feature of the lasso peptides of Tfus and Tcel is that they harbor the largest proteinogenic amino acid, Trp, as the first core residue (F g. 4A).
  • RODEO has identified cases where every residue except Pro is represented as the first residue of the core. Indeed, two cases were identified where Leu resides at this position (Fig. 3C).
  • TfusA was cloned into a pET28 derivative that provides an N-terminai maltose-binding protein (BP) tag, A second "Duet” piasmid harbored tfusC and tfusE-B.
  • BP N-terminai maltose-binding protein
  • "fusilassin” was detected by MALDI-TOF-MS (m/z 2289, Fig. 4B) after a methanolic extraction. After HPLC, high-resolution Orbitrap MS/MS analysis confirmed the identity of the peptide.
  • peptide is threaded, as opposed to a "branched cyclic" conformation, based on the following data: (i) resistance of centrally located amide NHs to deuterium exchange, (ii) thermal stability by HPLC after heat treatment (95 °C, 4 h), and (Hi) resistance to protease digestion.
  • Anecdotal evidence also supports the threaded state given that no lasso peptide has ever been isolated in the branched cyclic form. Fusilassin lasso sequences for Thermoactinomyces vulgaris and Thermobifida fusca YX were determined using RODEO. See Tietz et al.
  • a leader for Thermoactinomyces vulgaris was determined to be MEKQKETKKEYSSPRLIELGDIVEITF (SEQ ID NO: 10) and the core was determined to be GGKPGWGSDTYSQRYPRSDED (SEQ ID NO: 1 1 ).
  • a leader for Thermobifida fusca YX was determined to be MEKKKYTAPQLAKVGEFKEATG (SEQ ID NO: 12) and the core was determined to be WYTAEWGLELIFVFPRFI (SEQ ID NO: 13).
  • the proteins from Thermobifida fusca were cloned and obtained as full length and as a soluble protein so this gene cluster was targeted for heterologous expression in E. coil.
  • the pACYCDuef piasmid was used as the vector backbone for the biosynthetic gene cluster. Gibson assembly was used to clone 5'-TfusE-RBS-TfusB- 3' into MCS2 (primers: TfusB_F/R and TfusB_F/R). A ribosome binding site was inserted between the genes by adding the appropriate sequence into TfusE_R and TfusB_F. TfusC was then cloned into MCS1 using restriction enzyme digestion (primers: TfusC_F/R). See Table 1 .
  • the precursor peptide was cloned into a modified pET28 piasmid in frame with an N- terminal MBP tag. See Fig. 5.
  • the pET28 and pACYC plasmids were co-transformed into E. coii, which was grown to 0.8 ODeoo.
  • the plasmids were induced with 0.5 mM IPTG for 18 hours at 37°C. Ceil pellets were harvested and extracted with methanol.
  • the fusilassin lasso peptide was analyzed by MALDi-TOF-MS.
  • the MALDI-TOF-MS results are shown in Fig. 6.
  • Table 2 shows the protein name, molecular weight, and concentration of the protein purified from E. coii heterologous expression
  • the two plasmid method of production of lasso peptides was used to evaluate the biosynthetic tolerance of the fusilassin pathway (Fig. 1 1 ). in all other studied cases of lasso peptides, the first position of the core peptide was largely intolerant to substitution.
  • the two plasmid methodology was used to explore a subset of chemical space at position one of fusilassin. Substitution of the first position (Trp) of the fusilassin core peptide with Phe, Tyr, Lys, His, Ala and Leu resulted in cyclized fusilassin. See Fig. 1 1 .
  • Table 3 shows the reaction conditions for reconstituting the Tfus and Tcel lasso peptide biosynthetic systems.
  • A is the precursor peptide
  • B is the leader peptidase
  • C is the lasso cyclase
  • E is the RRE (RiPP) Recognition Element. The reactions were run for about 18 hours at room temperature.
  • Fusilassin has been reconstituted via in vitro biosynthesis at ⁇ mol% lasso cyclase, and at lasso peptide precursor amounts of up to 1 mmoi. Fusilassin has been produced at multi-milligram amounts using only the purified enzymes described above.
  • the RiPP Recognition Element is a domain of -90 aa with structural homology to PqqD.
  • Ubiquitous to lasso peptide BGCs Figs. 1 B, 3B
  • RREs are widely deployed in prokaryotic RiPP BGCs and govern the association between the leader peptide and biosynthetic protein(s).
  • RRE:leader binding and most dissociation constants are in the nM range.
  • a few RRE structures are available with each adopting a triple a-helix/p-sheet fold. Interestingly, the leader peptide binds as if if were the fourth cs-sheet.
  • nucleic acid molecules encoding a lasso precursor peptide, a solubility enhancing polypeptide, a lasso leader peptidase; a lasso cyclase; and a RiPP recognition element can be host cell "codon optimized", i.e., at least 50%, 60%, 70%, 75%, 80%, 85%, 90% of 95% of the codons of the nucleic acids are replaced with codons that encode the same amino acid but that are preferred by the host ceils, e.g., E. coli ceils, thus improving or optimizing expression in the E. coli cells.
  • a nucleic acid molecule is generated that alters "wild-type" codons to codons more frequently utilized by the host cell (e.g., E. coii).
  • TfusA that has been codon optimized for E. coii is operab!y linked to a solubility enhancing peptide (e.g., MBP) on a first piasmid and TfusC, TfusE, and TfusB can be present on a second piasmid. See Fig. 8. Both of these piasmids are used to transform a host cell.
  • solubility enhancing peptide e.g., MBP
  • a codon optimized lasso leader precursor e.g. TfusA
  • a solubility enhancing peptide e.g., MBP
  • Nucleic acid molecules encoding a lasso leader peptidase; a lasso cyclase; and a RiPP recognition element (RRE) e.g., TfusC, TfusE, and TfusB
  • RRE RiPP recognition element

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Abstract

L'invention concerne des procédés de reconstitution recombinants et in vitro pour la production de peptides lasso. L'invention concerne également des procédés de criblage de peptides lasso.
PCT/US2018/026101 2017-04-04 2018-04-04 Procédés de production de peptides lasso biologiquement actifs Ceased WO2018187482A1 (fr)

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WO2019191571A1 (fr) * 2018-03-30 2019-10-03 Lassogen, Inc. Procédés de production, de découverte et d'optimisation de peptides lasso
WO2020123387A1 (fr) * 2018-12-10 2020-06-18 Lassogen, Inc. Systèmes et procédés de découverte et d'optimisation de peptides lasso
US20220033446A1 (en) * 2018-12-10 2022-02-03 Lassogen, Inc. Systems and methods for discovering and optimizing lasso peptides
EP3908115A4 (fr) * 2018-12-10 2023-01-25 Lassogen, Inc. Systèmes et procédés de découverte et d'optimisation de peptides lasso
CN110343709A (zh) * 2019-05-17 2019-10-18 中国极地研究中心(中国极地研究所) 一种北极拟诺卡氏菌套索肽基因簇及其克隆与表达方法
CN110343709B (zh) * 2019-05-17 2022-11-15 中国极地研究中心(中国极地研究所) 一种北极拟诺卡氏菌套索肽基因簇及其克隆与表达方法
WO2021141901A1 (fr) * 2020-01-06 2021-07-15 Lassogen, Inc. Peptides lassos pour le traitement du cancer
EP4121547A4 (fr) * 2020-03-19 2024-07-03 Lassogen, Inc. Procédés et systèmes biologiques de découverte et d'optimisation de peptides lasso

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